in vitro node culture

Preparing the nodes

The advantage of this technique is that the new plants are clones of their parents and look like
they do. We have used this technique to propagate: Phalaenopsis, Doritis pulcherima, Phaius
tankervilleae and Chiloschista lunifera. Suitable are nodes, which you cut diagonal with 1 cm
below and above of the eye on the flower stalk. It is very important to us a very sharp knife
because otherwise the tissue will be hurt to much.

Phalaenopsis flower stalk with bract

Next you have to remove the bract covering the node carefully.

Phalaenopsis flower stalk (node) without bract

Which media should you use ?

To initiate the growth of the "sleeping eye" we have to use media which includes cytocinins
(phytohormon). We use Sigma´s P6793 (Phytotechlab P793).

Preparing the flasking area:

You can use the same equipment we discribed under Seed sowing.

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Flasking the nodes:
Dip the trimed nodes for a few seconds in 70% ethanol. After that put the nodes for 30 minutes
in 0,5% hydrogen peroxide (H2O2). Next put them for 15 minutes in 3% H2O2. When the 15
minutes elapsed, place the sterilized nodes (in the test tube) on the grill which is lying in the
steam (sterile area). Now, pick up a flask and open it in the steam. The cap should be placed on
the ethanol soaked kitchen paper. Take your forceps and pass the flame of your candle to
sterilize it. Transfer the forceps to the sterile area (steam) and catch one node which is swimming
in the hydrogen peroxide solution and stick it with the end at the bottom of the node into the
media.
Next, dip the forceps into the boilding water to remove all rests of media and transfer the forceps
into the bottle with 70% ethanol. Close the flask (in the steam) and placed it somewhere on you
desk for labeling. With the next flask you can process in the same way.

Hint: To make your sterilization solution more effective, add a drop of dish washing solution to
your hydrogen peroxide.

Further care:
The place where you culture your nodes should be bright and warm (about 20 °C). Prevent direct
sun because it will become to hot inside the flasks if they are standing in direct sun.

growing Chiloschista lunifera bud

Because of the size and the structure of the nodes the contamination rate is higher than using
asymb. seed germination. So, it´s very important to check them in the first week every day if
there are any contaminations. If you find some fungi or bacteria you can try to sterilize them
once again.

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Many nodes exudate phenolic compounds into media which make the media black. This phenolic
exudations will kill your nodes if you don´t replate them to new media. Many nodes stop
exudating phenolic compounds after 3 or 4 replatings.

phenolic exudations

As soon as the node has got 2 or 3 leaves you should replate it to media without hormones (e.g.
Sigma P6668) to initiate root development.

What can I do if I want more than one plant ?

If you want to produce more than one clone you should cut the top 1/3 of the node. This will
cause the node to put out up to a about dozen shoots instead of one.

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 Phaius tankervilleae node culture Phalaenopsis equestris young plant from a node

We use glass jars with a volume of about 350ml for replating. They are sealed with cling film.
More information can be found in our article "closing in vitro flasks with cling film".

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